Identification and Expression Analysis of SiSAP Gene Family in Foxtail Millet（Setaria italica）

doi:10.13560/j.cnki.biotech.bull.1985.2024-0576

Abstract

Abstract:

【Objective】Stress associated proteins（SAP）, a class of A20/AN1 zinc finger domain-containing proteins, play an critical role in plant resistance to abiotic stresses. However, the functions of SiSAP gene family in foxtail millet remain elusive. This study aims to identify the SiSAP gene family in foxtail millet and analyze its expression patterns, laying the foundation for exploring the function of SiSAP gene.【Method】The SiSAP gene family was identified and analyzed using bioinformatics approaches, and its expression levels under different abiotic stresses and hormone treatments were examined by real-time quantitative PCR（RT-qPCR）.【Result】The SiSAP gene family in foxtail millet（Setaria italica）consisted of 17 members, unevenly distributed on six chromosomes. Among these, 15 members had no introns, whereas the remaining contained one intron. The protein length, molecular weight, and isoelectric point ranged in 150-291 aa, 15.05-32.12 kD, and 6.62-9.36, respectively. Phylogenetic analysis revealed that the SiSAP gene family had a closer genetic relationship with monocots. The promoter regions of SiSAP gene family contained cis-acting elements involving in stresses and hormone responses, as well as transcription factor binding sites such as ERF, DOF, C2H2, etc. Transcriptome data indicated that nine genes, including SiSAP1, SiSAP2, SiSAP3, SiSAP4, SiSAP6, SiSAP7, SiSAP8, SiSAP11, and SiSAP14, were expressed in various tissues, while the remaining eight genes were almost not expressed. RT-qPCR analysis showed that the nine genes presented distinct response patterns to low temperature, high salt and drought stresses, as well as to treatments with abscisic acid, methyl jasmonate, auxin and gibberellin.【Conclusion】The SiSAP gene family may be involved in response to drought, salt and low temperature stresses in foxtail millet.

Key words: foxtail millet, SAP gene family, abiotic stress, hormone response, expression analysis

LI Yu-xin, LI Miao, DU Xiao-fen, HAN Kang-ni, LIAN Shi-chao, WANG Jun. Identification and Expression Analysis of SiSAP Gene Family in Foxtail Millet（Setaria italica）[J]. Biotechnology Bulletin, 2025, 41(1): 143-156.

Figures/Tables 13

Table 1 Primers for RT-qPCR in this study

基因名称Gene name	正向引物Forward primer（5'-3'）	反向引物Reverse primer（5'-3'）
SiSAP1	CTGCTGCTGTAATCCCCAAG	AGTGGCAGTCATGTTTGTCG
SiSAP2	GTTCCTCGCAATTTTCTGGA	CCAAGTACGCGTTTGCATAG
SiSAP3	ACGGTATATCTTGCGCTCGT	GCACTGTTGCCGAGATGTAA
SiSAP4	GCAGATAGCCAAGCAGAACC	CCACCACCTGAAGGAACCTA
SiSAP6	GTCTCCTCTCGCTCATCACG	CCTCACGCTTCTTCTTCTGC
SiSAP7	AAGATTCCATCCAGCAGAGC	CATCACCGCCATCATTACAC
SiSAP8	ATCCGGTGTTGCTAGTGGAG	ATGGGCAAATTTGAAGTCCA
SiSAP11	ATACCGGACCCAAACAACTG	ACCGTCTCAAGACAGGTTCG
SiSAP14	AACCCATTTCCTCTCCCTCA	GGCTCCTCCACCTTCTTGTC
SiACTIN	TGATCTCACTGACAGTCTGATG	GATGTCTCTTACAATTTCCCGC

Fig. 1 Location of SiSAP family members in foxtail millet in chromosome

Table 2 Identification of SiSAP gene family members

基因Gene	基因号Gene ID	基因位置Gene location	蛋白长度Protein length/aa	分子量Molecular weight/kD	等电点pI	亲水性GRAVY
SiSAP1	Seita.1G049700	Chr.1:4 826 713-4828307	172	18.38	7.99	-0.216
SiSAP2	Seita.1G050100	Chr.1:4 844 574-4846624	172	18.21	8.28	-0.198
SiSAP3	Seita.1G179600	Chr.1:25 817 455-25 819 730	150	15.65	9.28	-0.289
SiSAP4	Seita.2G045100	Chr.2:3 644 125-3 646 735	165	18.09	8.02	-0.479
SiSAP5	Seita.2G188600	Chr.2:28 469 651-28 470 203	184	19.74	8.75	-0.526
SiSAP6	Seita.2G195200	Chr.2:29 175 966-29 176 521	185	19.76	9.04	-0.546
SiSAP7	Seita.2G252300	Chr.2:35 515 181-35 516 761	170	17.55	8.90	-0.234
SiSAP8	Seita.2G358600	Chr.2:43 671 478-43 674 297	291	32.12	8.66	-0.627
SiSAP9	Seita.5G232000	Chr.5:29 446 450-29 446 969	173	17.47	8.96	-0.097
SiSAP10	Seita.5G232100	Chr.5:29 462 246-29 462 780	178	17.86	9.06	-0.063
SiSAP11	Seita.5G297700	Chr.5:35 289 769-35 291 603	154	15.86	8.74	-0.352
SiSAP12	Seita.5G330400	Chr.5:37 812 781-37 813 282	167	17.11	8.76	-0.089
SiSAP13	Seita.6G159700	Chr.6:28 237 350-28 238 434	256	26.19	8.47	-0.190
SiSAP14	Seita.6G203700	Chr.6:32 355 017-32 361 096	179	18.82	9.14	-0.357
SiSAP15	Seita.7G243600	Chr.7:30 408 518-30 409 232	236	24.02	8.60	-0.373
SiSAP16	Seita.9G061100	Chr.9:3 504 533-3 504 971	146	15.05	9.36	-0.312
SiSAP17	Seita.9G316800	Chr.9:36 489 146-36 489 611	155	16.52	6.62	-0.408

Fig. 2 Analysis of SiSAP gene family evolutionary tree, gene structure and motifs A: Phylogenetic relationship. B: Gene structure. C: Conserved motifs. Motif2 and motif14-motif7 are A20; motif3-motif1, motif5-motif1 and motif9-motif3-motif11 are AN1

Fig. 3 Alignment and analysis of sequences of SiSAP protein A20 and AN1 are highlighted with blue and red squares, respectively

Fig. 4 Collinearity analysis of SiSAP gene family

Table 3 Ka/Ks ratios of segmental duplicated gene pairs in the SiSAP gene family

同源基因 Homologous gene	非同义替换率（Ka） Non-synonymous substitution	同义替换率（Ks） Synonymous substitution	非同义替换率/同义替换率 Ka/Ks
SiSAP4-SiSAP16	0.41	1.21	0.34
SiSAP7-SiSAP14	0.20	0.47	0.42

Fig. 5 Collinearity analysis of SiSAP gene family members and SAP genes in Oraza sativa and Arabidopsis thaliana

Fig. 6 Phylogenetic tree of SAP gene family in plants Red stars: Setaria italica. Orange checks: Oraza sativa. Blue triangles: Triticum aestivum. Green circles: Hordeum vulgare. Yellow rects: Arabidopsis thaliana. Purple squares: Tamarix hispida

Fig. 7 Prediction of cis-acting element and transcription factor binding sites in the SiSAP gene family of foxtail millet A: Analysis of cis-acting element in promoter sequence of SiSAP family genes. The different intensity colors and numbers of the grid indicate the cis-acting element numbers in the SiSAP genes. B: The different colored histogram represents the sum of the cis-acting elements in each category. C: Only the top 15 TFs that bind to the promoter of SiSAP genes are shown here. The different intensity colors and numbers of the grid indicate the numbers of putative TF sites

Fig. 8 Tissue expression pattern analysis of SiSAP gene family in foxtail millet T1: Immature seed at S1. T2: Immature seed at S2. T3: Immature seed at S3. T4: Immature seed at S4. T5: Immature seed at S5. T6: Seeds germinated for 3 d. T7: Plant of one-tip-two-leaf. T8: Root at filling stage. T9: Neck-panicle-internodes at filling stage. T10: Stem-top-second at filling stage. T11: Leaves after 2 d of heading（Leaf-top-2-3）. T12: Flag leaf at filling stage. T13: Leaf-top-fourth at filling stage. T14: Flag leaf sheath at filling stage. T15: Leaf-sheath-top-fourth at filling stage. T16: Primary panicle at differentiation stage. T17: Third panicle at differentiation stage. T18: Immature spikelet at S2. T19: Immature spikelet at S4

Fig. 9 Expression patterns of SiSAP gene in response to abiotic stress Abscissa indicates 0, 1, 3, 6 and 12 h after treatment. *, **, *** indicate P < 0.05, P < 0.01 and P < 0.001, respectively. The same below

Fig. 10 Expression patterns of SiSAP gene in response to hormone treatment

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